Over the current reporting period we published 3 major papers related to this project: (1) a paper in Nature describing the role of the calcium sensing receptor in the regulation of the NLRP3 inflammasome. The NLRP3 gene is mutated in 3 dominantly-inherited autoinflammatory diseases; (2) a paper in the Annals of Rheumatic Diseases describing a gene expression pattern associated with one of the autoinflamatory diseases caused by NLRP3 mutations; and (3) a paper in the American Journal of Human Genetics describing APLAID, a new dominantly-inherited autoinflammatory disease. Regulation of the NLRP3 Inflammasome Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as the cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1beta (IL-1beta) by activating caspase-1. Although several models for inflammasome activation, such as potassium efflux, generation of reactive oxygen species, and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. In this project we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular calcium and decreased cellular cyclic AMP (cAMP). Calcium or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyzes inositol-1,4,5-triphosphate production and thereby induces release of calcium from endoplasmic reticulum stores. The increased cytoplasmic calcium promotes the assembly of inflammasome components, and intracellular calcium is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1beta production from CAPS patients peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that calcium and cAMP are two key molecular regulators of the NLRP3 inflamasome that have critical roles in the molecular pathogenesis of CAPS. Gene Expression Profiling in CAPS Patients Treated with Anakinra The objective of this study was to analyze gene expression patterns and to define a specific gene expression signature in patients with the severe end of the spectrum of CAPS. The molecular consequences of IL-1 inhibition were examined by comparing gene expression patterns in 16 CAPS patients before and after treatment with anakinra. We collected peripheral blood mononuclear cells (PBMCs) from 22 CAPS patients with active disease and 14 healthy children. Transcripts that passed stringent filtering criteria (false discovery rate of less than 1%) were considered differentially expressed genes (DEGs). A set of DEGs was validated by quantitative RT-PCR and functional studies with primary cells from CAPS patients and healthy controls. We used 17 CAPS and 66 non-CAPS patient samples to create a set of gene expression models that differentiates CAPS patients from controls and from patients with other autoinflammatory conditions. Many of the DEGs are related to the regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion, and motility, and were in common with other systemic inflammatory diseases such as systemic-onset juvenile idiopathic arthritis. A set of gene expression-based models comprising the CAPS-specific gene expression signature correctly classified all 17 samples from an independent dataset. This classifier also correctly identified 15 of 16 post-anakinra CAPS patients despite the fact that these CAPS patients were in clinical remission, suggesting incomplete suppression of inflammation at these doses of anakinra. Overall, the data delineate a gene expression signature that clearly distinguishes CAPS patients from controls. Discovery of APLAID, an Autoinflammatory Disease Caused by a Hypermorphic Missense Mutation in PLCG2 Whole-exome sequencing was performed in a family affected by dominantly-inherited inflammatory disease characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. Exome data from 3 samples, including the affected father and daughter and unaffected mother, were filtered for the exclusion of reported variants, along with benign variants, as determined by PolyPhen-2. A total of 8 transcripts were identified as possible candidate genes. We confirmed a variant, p.Ser707Tyr, within PLCG2 as the only de novo variant that was present in two affected family members and not present in four unaffected members. PLCG2 encodes phospholipase Cgamma2 (PLCgamma2), an enzyme with a critical regulatory role in various immune and inflammatory pathways. The p.Ser707Tyr substitution is located in an autoinhibitory SH2 domain that is critical for PLCgamma2 activation. Overexpression of the altered p.Ser707Tyr protein and ex vivo experiments using affected individuals leukocytes showed clearly enhanced PLCgamma2 activity, suggesting increased intracellular signaling in the PLCgamma2-mediated pathway. Recently, our laboratory identified in individuals with cold-induced urticaria and immune dysregulation PLCG2 exon-skipping mutations resulting in protein products with constitutive phospholipase activity but with reduced intracellular signaling at physiologic temperatures. In contrast, the p.Ser707Tyr substitution in PLCgamma2 causes a distinct inflammatory phenotype that is not provoked by cold temperatures and that has different end-organ involvement and increased intracellular signaling at physiologic temperatures. Our results highlight the utility of exome-sequencing technology in finding causal mutations in nuclear families with dominantly-inherited traits otherwise intractable by linkage analysis. We have proposed the acronym APLAID (autoinflammatory PLCgamma2 associated antibody deficiency and immune dysregulation) to denote this clinically and immunologically distinct disease.